Synthesis of [18F]SU11248, a new potential PET tracer for imaging cancer tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2005.06.038

N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-[¹⁸F]fluoro-2-oxo-1, 2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide, a new potential positron emission tomography tracer for imaging cancer tyrosine kinase, has been prepared by the nucleophilic substitution of the nitro-precursor N-[2-(diethylamino)ethyl]-5-[(Z)-(5-nitro-2-oxo-1,2-dihydro-3H- indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide with K ¹⁸F/Kryptofix 2.2.2 followed by a simple chromatography methodology combined solid-phase extraction with high-performance liquid chromatography purification procedures in 15-25% radiochemical yields.

Full text of DOI:10.1016/j.bmcl.2005.06.038

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4380–4384
Synthesis of [18F]SU11248, a new potential PET tracer
for imaging cancer tyrosine kinase
Ji-Quan Wang,a Kathy D. Miller,b George W. Sledgeb and Qi-Huang Zhenga,*
aDepartment of Radiology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
bDepartment of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
Received 6 May 2005; revised 8 June 2005; accepted 9 June 2005
Available online 12 July 2005
Abstract—N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-[18F]fluoro-2-oxo-1, 2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-
3-carboxamide, a new potential positron emission tomography tracer for imaging cancer tyrosine kinase, has been prepared by
the nucleophilic substitution of the nitro-precursor N-[2-(diethylamino)ethyl]-5-[(Z)-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-
ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide with K18F/Kryptofix 2.2.2 followed by a simple chromatography method-
ology combined solid-phase extraction with high-performance liquid chromatography purification procedures in 15–25%
radiochemical yields.
ꢀ 2005 Elsevier Ltd. All rights reserved.
N-[2-(Diethylamino)ethyl]-5-[(Z)-(5-fluoro-2-oxo-1,2-di-
hydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyr-
role-3-carboxamide (SU11248) is a novel tyrosine kinase
inhibitor developed by SUGEN, which targets both vas-
cular endothelial growth factor (VEGF) and plated-de-
rived growth factor (PDGF) receptor tyrosine kinases
(RTKs) and has been entered to clinical trials as an anti-
tumor drug for the treatment of cancers.1 The overexpres-
sion of the RTKs in tumors indicates that the RTKs are
the suitable target for tumor imaging agent development.
Positron emission tomography (PET) is a functional bio-
medical imaging modality that can probe tumor cell phys-
iology. PET and a positron-labeled tyrosine kinase
inhibitor that inhibits selectively to RTKs may prove to
be a useful tool for monitoring RTK levels in tumor tis-
sues and for evaluating the effectiveness of the antitumor
drug. To further develop therapeutic drug SU11248 as
a diagnostic agent, we report here the design and
synthesis of N-[2-(diethylamino)ethyl]-5-[(Z)-(5-[18F]
fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,
4-dimethyl-1H-pyrrole-3-carboxamide ([18F]SU11248)
through a simple chromatography-methodology-com-
bined solid-phase extraction (SPE) and high-performance
liquid chromatography (HPLC) purification procedures
for the first time.
The synthesis of the precursor N-[2-(diethylamino)
ethyl]-5-[(Z)-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-yl-
idene) methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide
(9) and reference standard SU11248 (1) was performed
using a modification of the literature procedure.1,2 The
synthetic approach is outlined in Scheme 1. Treatment
of diketene (2) with N,N-diethylethylenediamine in
tert-butyl methyl ether furnished N-[2-(diethylami-
no)ethyl]-3-oxobutanamide (4) in excellent yield
(ꢀ100%). The b-ketoamide 4 was prone to decomposi-
tion and, therefore, had to be used immediately. Oxime
tert-butyl 2-(hydroxyimino)-3-oxo-butanoate (5) de-
rived from tert-butyl acetoacetate (3) in quantitative
yield was treated with amide 4 in the presence of zinc
and acetic acid according to the classic Knorr pyrrole
formation conditions, which led to pyrrole tert-butyl
4-[[[2-(diethylamino)ethyl]amino]carbonyl]-3,5-dimeth-
yl-1H-pyrrole-2-carboxylate (6) in 58% yield. The decar-
boxylation of pyrrole 6 under acidic conditions (H2SO4/
MeOH) provided the decarboxylated product N-[2-
(diethylamino)ethyl]-2,4-dimethyl-1H-pyrrole-3-carbox-
amide (7) in 87% yield. The a-free pyrrole 7 was treated
with chloromethylenedimethylammonium chloride in
acetonitrile to form the Vilsmeier–Haack adduct 8 in
situ.2 Addition of 5-fluorooxindole and KOH to the
reaction mixture at this stage through Eschenmoser type
condensation afforded the reference standard SU11248
(1) in 54% yield. Similarly, addition of 5-nitrooxindole
and KOH to the reaction mixture afforded the nitro-pre-
cursor (9) in 61% yield. The overall chemical yields for
Keywords: [18F]SU11248; Synthesis; Positron emission tomography;
Tracer; Tyrosine kinase; Cancer.
*
Corresponding author. Tel.:+1 317 278 4671; fax: +1 317 278 9711;
e-mail: qzheng@iupui.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.038

J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4380–4384
4381
Scheme 1. Synthesis of SU11248 (1) and nitro-precursor (9).
the reference standard 1 and nitro-precursor 9 in five
steps are moderate.
15% MeOH/CH2Cl2 and the solvent was evaporated un-
der high vacuum to give the mixture residue of the pre-
cursor and product, and then eluted with 2/8/90 HOAC/
EtOH/H2O to desalt K18F and remove the Kryptofix
2.2.2, so that the SiO2 Sep-Pak cartridge can be repeat-
edly used in the tracer production. The existence of the
catalyst Kryptofix 2.2.2 and nonreacted K18F would af-
fect the purification of labeled product from its mixture
with precursor and the quality control of the tracer pro-
duction;12 therefore, they needed to be removed before
[18F]1 was separated from the mixture. The SPE tech-
nique reduces the tasks and complexity prior to HPLC
purification of the radiolabeling mixture. Then, the mix-
ture containing precursor and product was diluted with
HPLC mobile phase and loaded on the HPLC column
and purified by semipreparative HPLC method. The
retention time (RT) difference between target tracer
[18F]1 and the precursor 9 in the preparative HPLC
system we used was ꢀ1.7min, which warranted the effi-
cient purification of the target tracer from unlabeled
precursor. The nitro-precursor has similar structure with
the final fluoro-tracer, they may have similar biological
activity, and the precursor would affect the PET studies
of the tracer. Therefore, the precursor needed to be re-
moved before [18F]1 was formulated in saline as final
The nitro-precursor 9 was labeled by a conventional
nucleophilic substitution3–7 with K18F/Kryptofix 2.2.2
in acetonitrile at 120 ꢁC for 15–20 min to provide target
tracer [18F]SU11248 ([18F]1). The synthetic approach is
indicated in Scheme 2. The radiolabeling reaction is a
key step in the radiosynthesis of final compound. In this
reaction, Kryptofix 2.2.2 was used as a phase transfer
catalyst, which catalyzed the fluorination of nitro-pre-
cursor. A novel chromatography-methodology-com-
bined SPE with HPLC techniques was employed in the
isolation and purification of the target tracer from
radiolabeling reaction mixture. A flow chart for the iso-
lation procedure is shown in Scheme 2. The radiolabel-
ing mixture was passed through a Silica Sep-Pak to
remove Kryptofix 2.2.2 and nonreacted K18F. The large
polarity difference between nitro-precursor, labeled
product, Kryptofix 2.2.2, and nonreacted K18F permit-
ted the use of a simple SPE technique8–11 for fast isola-
tion of nitro-precursor and labeled product from the
radiolabeling reaction mixture. The key part in this tech-
nique is a SiO2 Sep-Pak type cartridge that contains
ꢀ0.5–2 g of adsorbent. The Sep-Pak was eluted with

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J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4380–4384
Scheme 2. Synthesis, isolation, and purification of [18F]SU11248 ([18F]1).
pure product for further study. The radiochemical yield
of [18F]1 was 15–25% at the end of bombardment
(EOB).
istry results provide the foundation for further evaluation
of the tracer [18F]SU11248 as a new potential PET tumor
imaging agent for tyrosine kinase in vivo.
The application of combined chromatography method-
ology using both SPE and HPLC techniques simplified
the isolation and purification procedures of the radio-
synthesis of [18F]SU11248, so that it will be amenable
for automation.5 The optimization through SPE and
HPLC methods for the tracer [18F]SU11248 production
provides a general and useful methodology, which could
be used to purify all fluorine-18 PET radiotracers pre-
pared by nucleophilic substitution of the precursors
radiolabeling with K18F/Kryptofix 2.2.2.
Acknowledgments
This work was partially supported by the Susan G. Ko-
men Breast Cancer Foundation Grants IMG 02-1550
and BCTR0504022 (to Q.-H.Z.), the Breast Cancer Re-
search Foundation (to K.D.M. and G.W.S.), and the
Indiana Genomics Initiative (INGEN) of Indiana Uni-
versity, which is supported in part by Lilly Endowment
Inc. The refereesÕ criticisms and editorÕs comments for
the revision of the manuscript are greatly appreciated.
The experimental details are given for the new com-
pound nitro-precursor 9 and tracer [18F]1, and only
characterization data are given for other known com-
pounds 4–7 and 1.13
References and notes
1. Sun, L.; Liang, C.; Shirazian, S.; Zhou, Y.; Miller, T.; Cui,
J.; Fukuda, J. Y.; Chu, J. Y.; Nematalla, A.; Wang, X.;
Chen, H.; Sistla, A.; Luu, T. C.; Tang, F.; Wei, J.; Tang,
C. J. Med. Chem. 2003, 46, 1116.
2. Manley, J. M.; Kalman, M. J.; Conway, B. G.; Ball, C. C.;
Havens, J. L.; Vaidyanathan, R. J. Org. Chem. 2003, 68,
6447.
3. Fei, X.; Zheng, Q.-H.; Wang, J.-Q.; Stone, K. L.;
Martinez, T. D.; Miller, K. D.; Sledge, G. W.; Hutchins,
G. D. Bioorg. Med. Chem. Lett. 2004, 14, 1247.
4. Fei, X.; Wang, J.-Q.; Miller, K. D.; Sledge, G. W.; Hutchins,
G. D.; Zheng, Q.-H. Nucl. Med. Biol. 2004, 31, 1033.
In conclusion, a novel and general chromatography-
methodology-combined SPE with HPLC techniques has
been well developed, and the detailed isolation and puri-
fication procedures used in the radiosynthesis of
[18F]SU11248 have been described. The synthetic proce-
dures that provide nitro-precursor and reference standard
SU11248 also have been developed in this regard. These
methods are efficient and convenient, and work very well
for the routine production of a variety of other fluorine-18
PET tracers used in our PET imaging center. These chem-

J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4380–4384
4383
5. Wang, J.-Q.; Zheng, Q.-H.; Fei, X.; Mock, B. H.;
Hutchins, G. D. Bioorg. Med. Chem. Lett. 2003, 13, 3933.
6. Wang, J.-Q.; Zheng, Q.-H.; Fei, X.; Liu, X.; Gardner, T.
A.; Kao, C.; Raikwar, S. P.; Glick-Wilson, B. E.; Sullivan,
M. L.; Mock, B. H.; Hutchins, G. D. Synth. Commun.
2004, 34, 917.
7. Zheng, Q.-H.; Wang, J.-Q.; Liu, X.; Fei, X.; Mock, B. H.;
Glick-Wilson, B. E.; Sullivan, M. L.; Raikwar, S. P.;
Gardner, T. A.; Kao, C.; Hutchins, G. D. Synth. Commun.
2004, 34, 689.
8. Mulholland, G. K.; Zheng, Q.-H.; Mock, B. H.; Vavrek,
M. T. J. Label. Compd. Radiopharm. 1999, 42, S459.
9. Wang, J.-Q.; Miller, M. A.; Fei, X.; Stone, K. L.;
Lopshire, J. C.; Groh, W. J.; Zipes, D. P.; Hutchins, G.
D.; Zheng, Q.-H. Nucl. Med. Biol. 2004, 31, 957.
10. Zheng, Q.-H.; Stone, K. L.; Mock, B. H.; Miller, K. D.;
Fei, X.; Liu, X.; Wang, J.-Q.; Glick-Wilson, B. E.; Sledge,
G. W.; Hutchins, G. D. Nucl. Med. Biol. 2002, 29, 803.
11. Zheng, Q.-H.; Liu, X.; Fei, X.; Wang, J.-Q.; Mock, B. H.;
Glick-Wilson, B. E.; Sullivan, M. L.; Hutchins, G. D.
Bioorg. Med. Chem. Lett. 2003, 13, 1787.
J = 7.4 Hz, CH2CH3). (f) Compound 1: an orange solid in
54% yield, Rf = 0.47 (46:4:1, CH2Cl2/MeOH/NH4OH), mp
215–217 ꢁC. 1H NMR (300 MHz, CDCl3): d 13.31 (s,
1H, NH), 8.90 (s, 1H, NH), 7.22 (s, 1H, olefinic H),
7.14 (dd, 1H, J1 = 8.8 Hz, J2 = 2.2 Hz, phenyl H), 6.85
(td, 1H, J1 = 8.8 Hz, J2 = 2.2 Hz, phenyl H), 6.78 (dd, 1H,
J1 = 8.1 Hz, J2 = 4.4 Hz, phenyl H), 6.72 (t, 1H,
J = 4.4 Hz, CONHCH2), 3.54 (q, 2H, J = 5.2 Hz,
HNCH2CH2N), 2.71 (t, 2H, J = 5.9 Hz, HNCH2CH2N),
2.64 (q, 4H, J = 6.6 Hz, NCH2CH3), 2.55 (s, 3H, ring-
CH3), 2.31 (s, 3H, ring-CH3), 1.05 (t, 6H, J = 7.4 Hz,
NCH2CH3). (g) Compound 9: chloromethylenedimethyl-
ammonium chloride (1.70 g, 13.28 mmol) was added to a
250-mL two-necked flask equipped with an addition
funnel and a nitrogen inlet. CH3CN (20 mL) was added
dropwise via the addition funnel to the flask. The pyrrole 7
(2.98 g, 12.56 mmol) was dissolved in CH3CN (25 mL)
and added to the flask through the addition funnel. The
amide chloride gradually dissolved and the reaction
solution turned dark orange. After 15 min, an orange
solid precipitated out of the solution. The reaction was
completed in 40 min. Then, to the above flask were added
5-nitrooxindole (2.0 g, 13.24 mmol) and powdered KOH
(2.59 g, 56.11 mmol). The mixture was stirred at room
temperature for 2 days. An orange precipitate was filtered,
washed with water, and CH3CN to give an orange solid 9
(3.24 g, 61%), mp 265–266 ꢁC. 1H NMR (300 MHz,
DMSO-d6): d 13.95 (s, 1H, NH), 11.90 (s, 1H, NH), 9.24
(d, 1H, J = 2.2 Hz, phenyl H), 8.46 (dd, 1H, J1 = 8.8 Hz,
J2 = 2.2 Hz, phenyl H), 8.41 (s, 1H, olefinic H), 7.90 (t,
1H, J = 5.2 Hz, CONHCH2), 7.44 (d, 1H, J = 8.8 Hz,
phenyl H), 3.65–3.80 (m, 4H, NHCH2CH2N), 2.95 (q, 4H,
J = 5.9 Hz, NCH2CH3), 2.88 (s, 3H, ring-CH3), 2.87 (s,
3H, ring-CH3), 1.39 (t, 6H, J = 7.4 Hz, NCH2CH3).
LRMS (CI, m/z): 236 (100%), 426 [(M+H)+, 8.2%].
HRMS (CI, m/z): calcd for C22H28N5O4: 426.2141, found:
426.2141. (h) Tracer [18F]1: no-carrier-added aqueous
H18F (0.5 mL) prepared by 18O(p,n)18F nuclear reaction
in a RDS-112 cyclotron on an enriched H218O water
(95+%) target was added to a Pyrex vessel that contains
K2CO3 (4 mg, in 0.2 mL H2O) and Kryptofix 2.2.2 (10 mg,
in 0.5 mL CH3CN). Azeotropic distillation at 115 ꢁC with
HPLC grade CH3CN (3· 1 mL) under a nitrogen steam
efficiently removed water to form anhydrous K18F–
Kryptofix 2.2.2 complex. The nitro-precursor 9 (2–3 mg,
dissolved in 0.5 mL CH3CN) was introduced to the K18F–
Kryptofix 2.2.2 complex. The radiolabeling reaction was
monitored by analytical radio-HPLC method, in which we
employed a Prodigy (Phenomenex) 5 lm C-18 column,
4.6 · 250 mm; 3:1:1, CH3CN/MeOH/20 mM, pH 6.7
12. Mock, B. H.; Winkle, W.; Vavrek, M. T. Nucl. Med. Biol.
1997, 24, 193.
13. Experimental details and characterization data. (a) Gen-
eral: all commercial reagents and solvents were used
without further purification. Melting points were deter-
mined on a MEL-TEMP II capillary tube apparatus and
were uncorrected. 1H NMR spectra were recorded on a
Bruker QE 300 NMR spectrometer using tetramethyl-
silane (TMS) as an internal standard. Chemical shift data
for the proton resonances were reported in parts per
million (d) relative to internal standard TMS (d 0.0). Low-
resolution mass spectra were obtained using a Bruker
Biflex III MALDI-TOF mass spectrometer, and high-
resolution mass measurements were obtained using a
Kratos MS80 mass spectrometer, in the Department of
Chemistry at Indiana University. Chromatographic sol-
vent proportions are expressed on a volume:volume basis.
Thin-layer chromatography was run using Analtech silica
gel GF uniplates (5 · 10 cm). Plates were visualized by UV
light. Normal phase flash chromatography was carried out
on EM Science silica gel 60 (230–400 mesh) with a forced
flow of the indicated solvent system in the proportions
described below. All moisture-sensitive reactions were
performed under a positive pressure of nitrogen main-
tained by a direct line from a nitrogen source. Sterile
Millex-GS 0.22-lm vented filter unit was obtained from
Millipore Corporation (Bedford, MA, USA). (b) Com-
pound 4: compound 2 and tert-butyl methyl ether were
reacted with N,N-diethylethylenediamine to give a brown
liquid 4 in 100% yield, which was used directly in next step
without further purification. (c) Compound 5: the reaction
of compound 3 and acetic acid with a solution of NaNO2
in water gave product oxime 5 in quantitative yield, and
the solution was used directly in next step without further
purification. (d) Compound 6: an off-white solid in 58%
yield, Rf = 0.70 (46:4:1, CH2Cl2/MeOH/NH4OH). 1H
NMR (300 MHz, CDCl3): d 9.15 (br s, 1H, NH), 6.41
(br s, 1H, NH), 3.45 (q, 2H, J = 5.9 Hz, HNCH2CH2N),
2.62 (t, 2H, J = 5.9 Hz, HNCH2CH2N), 2.54 (q, 4H,
J = 7.4 Hz, CH2CH3), 2.49 (s, 3H, ring-CH3), 2.48 (s, 3H,
ring-CH3), 1.57 (s, 9H, t-Bu), 1.01 (t, 6H, J = 7.4 Hz,
CH2CH3). (e) Compound 7: a viscous brown oil in 87%
yield, Rf = 0.62 (46:4:1, CH2Cl2/MeOH/NH4OH). 1H
NMR (300 MHz, CDCl3): d 8.53 (br s, 1H, NH), 6.45
(br s, 1H, NH), 6.34 (s, 1H, olefinic H), 3.45 (q, 2H,
J = 5.1 Hz, HNCH2CH2N), 2.61 (t, 2H, J = 5.9 Hz,
HNCH2CH2N), 2.54 (q, 4H, J = 7.4 Hz, CH2CH3), 2.47
(s, 3H, ring-CH3), 2.24 (s, 3H, ring-CH3), 1.01 (t, 6H,
À
KHPO4 mobile phase, 1.5 mL/min flow rate, UV
(240 nm) and c-ray (NaI) flow detectors. Retention times
(RTs) in the analytical HPLC system were:
RT9 = 2.83 min, RT[18F]1 = 3.66 min, and RTK18F =
1.88 min. The reaction mixture was sealed and heated at
120 ꢁC for 15–20 min and was subsequently allowed to
cool down, at which time the crude product was passed
through a Silica Sep-Pak cartridge (Waters Corporate
Headquarters, Milford, MA, USA) to remove Kryptofix
2.2.2 and unreacted K18F. The Sep-Pak column was eluted
with 15% MeOH/CH2Cl2 (5.0 mL), and the fractions were
passed onto a rotatory evaporator. The solvent was
removed by evaporation under high vacuum (0.1–
1.0 mmHg) to give a crude product [18F]1. The mixture
containing precursor and product was purified with
semipreparative HPLC method, in which we employed a
Prodigy (Phenomenex) 5 lm C-18 column, 10 ·À250 mm;
3:1:1 CH3CN/MeOH/20 mM, pH 6.7 KHPO4 mobile
phase, 5.0 mL/min flow rate, UV (240 nm), and c-ray

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J.-Q. Wang et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4380–4384
(NaI) flow detectors. The contents of the mixture residue
70 min from EOB. RTs in the semipreparative HPLC
system were: RT9 = 5.03 min and RT[18F]1 = 6.74 min.
The radiochemical yield of [18F]1 was 15–25%. Chemical
purity, radiochemical purity, and specific radioactivity
were determined by analytical HPLC method. The
chemical purities of precursor 9 and standard sample 1
were >96%, the radiochemical purity of target radio-
tracer [18F]1 was >99%, and the chemical purity of
radiotracer [18F]1 was ꢀ93%. The specific radioactivities
of radiotracer [18F]1 were 0.8–1.2 Ci/lmol (n = 3–5) at
end of synthesis.
were diluted with HPLC mobile phase 3:1:1 CH3CN/
MeOH/20 mM, pH 6.7 KHPO4À, and injected onto the
semipreparative HPLC column. The product fraction was
collected, the solvent was removed by rotatory evapora-
tion under vacuum, and the final product [18F]1 was
formulated in saline, sterile-filtered through a sterile
vented Millex-GS 0.22-lm cellulose acetate membrane
and collected into a sterile vial. Total radioactivity was
assayed and total volume was noted. The overall
synthesis, purification, and formulation time was 60–